Biotechnological methods are used to an increasing extent in the investigation of proteins, peptides, nucleic acids and other biological entities. Due to its versatility and sensitivity to the entities, chromatography is often the preferred purification method in this context. The chromatography is based on principle of separating mixture by dissolving a mixture in a mobile phase (e.g. liquids like buffers) and passing it through a stationary phase (e.g., chromatography matrix that could be beads/resins). More specifically, a mixture which includes the target entity (e.g., a chemical/biological molecule) is introduced into a mobile phase, the mobile phase is then contacted with a stationary phase, also known as the chromatography matrix. The target entity will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the stationary matrix by the mobile phase. The interactions exploit differences in the physical or chemical properties of the target entity and other components in the mixture.
Chromatographic methods can be run in different modes of operation. The simplest mode is batch chromatography, wherein the mobile phase is added to a vessel containing stationary phase; interaction between target entity and stationary phase is allowed for a suitable period of time; the mobile phase is withdrawn; and an eluent is added to release the target entity. In column chromatography on the other hand, a mobile phase containing the sample (mixture) is added to the top of a column containing stationary phase. By opening up an outlet at the lower end of the column, the mobile phase is passed through the column, during which the sample interacts with the stationary phase. Elution is commonly performed by applying a small amount of eluent at the top, and again allowing the eluent to pass through the column either by gravity or using a chromatography system.
Once a suitable load of target entity has been obtained on the stationary phase, the liquid flow is changed from mobile phase to an eluent, optionally with one or more intermediate washings, and the target fraction is recovered from the eluent at the outlet of the column. The eluent will commonly comprise a gradient, such as a salt or pH gradient. To avoid contamination of large contaminants, and to obtain an advantageous liquid distribution throughout the column, the inlet is usually equipped with a filter and mechanical liquid distributor means. Most commonly, the outlet will similarly present both a filter and some mechanical liquid distributor means.
Many researchers who perform protein purification uses either a basic chromatography system or by manual methods. Due to the workflow compulsion, most of these users resort to self-packed empty columns, packing their own column with the media of their choice. A number of vendors offer empty columns in the field. Bio-Rad provides a low pressure chromatography column under the Econo brand. These are glass columns with flow adaptors, and withstand a low operating pressure of less than 1 bar. Pall Corporation provides LRC columns which can withstand an operating pressure of 10-30 bars. Their screw-lock system allows rapid coarse adjustment followed by precise fine adjustment of the piston (which gives coverage of the various bed heights).
Users who choose to pack their own columns face constant challenges in terms of limited pressure specification in low pressure column segment, affordability, ease of use, complex designs of endcap with adaptors, and limitation in the size/scale of columns. There is a need for better chromatography column design that is more user friendly and can withstand higher column pressure.